Persistence of the historical lineage I of West Africa against the ongoing spread of the Asian lineage of peste des petits ruminants virus

Abstract Peste des petits ruminants (PPR) is a highly contagious disease of small ruminants. The causal agent, PPR virus (PPRV), is classified into four genetically distinct lineages. Lineage IV, originally from Asia, has shown a unique capacity to spread across Asia, the Middle East and Africa. Recent studies have reported its presence in two West African countries: Nigeria and Niger. Animals are frequently exchanged between Mali and Niger, which could allow the virus to enter and progress in Mali and to other West African countries. Here, PPRV samples were collected from sick goats between 2014 and 2017 in both Mali and in Senegal, on the border with Mali. Partial PPRV nucleoprotein gene was sequenced to identify the genetic lineage of the strains. Our results showed that lineage IV was present in south‐eastern Mali in 2017. This is currently the furthest West the lineage has been detected in West Africa. Surprisingly, we identified the persistence at least until 2014 of the supposedly extinct lineage I in two regions of Mali, Segou and Sikasso. Most PPRV sequences obtained in this study belonged to lineage II, which is dominant in West Africa. Phylogenetic analyses showed a close relationship between sequences obtained at the border between Senegal and Mali, supporting the hypothesis of an important movement of the virus between the two countries. Understanding the movement of animals between these countries, where the livestock trade is not fully controlled, is very important in the design of efficient control strategies to combat this devastating disease.


| INTRODUC TI ON
Peste des petits ruminants (PPR) is a highly acute contagious transboundary infectious disease of small ruminants, primarily sheep and goats. It is one of the most economically important animal diseases in areas that rely on small ruminants for their livelihoods. The main clinical signs of PPR are the rapid rise in body temperature, the appearance of nasal and ocular secretions which become mucopurulent, necrotizing stomatitis, profuse, haemorrhagic diarrhoea in cases of parasitism and death. Signs of pneumonia (cough, dyspnoea), abortions that can be observed among pregnant animals, are important clinical aspects of PPR; because of its high contagiousness, high morbidity and mortality, PPR is a World organization for Animal Health (OIE) notifiable disease and since 2015 targeted for global eradication by 2030 (FAO/OIE, 2015).
Peste des petits ruminants is endemic in large parts of Africa, the Middle East and Asia . PPR is caused by peste des petits ruminants virus (PPRV) a member of the Morbillivirus genus in the Paramyxoviridae family [species: Small ruminant morbillivirus; (Amarasinghe et al., 2017)]. PPRV is a negative sense single-stranded ribonucleic acid (RNA) virus with an envelope varying in shape and size, ranging from 170 nm to 500 nm (Bourdin & Laurent-Vautier, 1967;Durojaiye et al., 1985). The PPRV genome has a length between 15,948 pb and 15,954 pb and encodes six structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), haemagglutinin protein (H) and a viral RNA-dependent polymerase (L) and two non-structural proteins C and V (Parida et al. 2015).
The PPR virus strains are divided into four genetic lineages (I, II, III and IV) based on N and F genes partial sequences (Kwiatek et al., 2007;Shaila et al., 1989). All PPRV lineages are circulating in the African continent: I and II in West Africa, III in East Africa and IV in Central, East, North and West Africa (Baazizi et al., 2017;Banyard et al., 2010;Dundon et al., 2020;Kwiatek et al., 2011;Muniraju et al., 2014). The lineages II and IV have the widest distribution, appearing to have replaced lineage I and III in West and East Africa, respectively (Dundon et al., 2020). Notably, the last confirmed presence of the lineage I in West Africa was in Senegal in 1994 and in Niger in 2001 (Tounkara et al., 2018). Lineage II PPR strains are now widespread across West Africa, probably linked with extensive transboundary animal movements (Tounkara et al., 2018). Strains of lineage IV have also started to emerge in West Africa in recent years, with a wide diversity of strains already described in Nigeria (Mantip et al., 2016;Woma et al., 2016), and a first description in Niger in 2013 (Souley et al., 2019;Tounkara et al., 2018). The lineage may have already spread further westward with the commercial movement of infected animals, but data are currently lacking to evaluate this risk.
The objective of this study is to undertake molecular epidemiol- spread to other West African countries. We explored this hypothesis by carrying out sampling at major crossing points at the border between Mali and Senegal, a major endpoint for livestock in West Africa. Nevertheless, the tissues for this study were collected respecting animal welfare. The study focussed on animals in outdoor environment where PPR is endemic. Animals showing symptoms corresponding to PPR infection were restrained to obtain ocular and nasal swabs.

| Ethical approval, sampling and RNA extraction
The tissue samples (lung, lymph nodes and/or spleen) were collected from animals, who died from infection or humanely killed after developing acute PPR symptoms, including mucopurulent nasal/ocular discharges, fever, diarrhoea, respiratory distress and loss of weight.
Samples were stored at 4°C during their transfer to the central veterinary laboratories and sent to CIRAD Montpellier France for processing in a Biosafety Level 3 containment Laboratory.
In the laboratory, the tissue samples were cut into small pieces and put in 15-ml tube then vortexed with 3 ml of Minimum essential Medium (MEM) (Invitrogen) and 2 mm glass beads. The swabs were processed in 1 ml of MEM and vortexed. All the sample suspensions were then centrifuged for 3 min at 1,000 g and the supernatant collected. RNA extraction was carried out using the NucleoSpin

| Sequencing and phylogenetic analysis
Positive PCR products were cleaned up and sequenced in both forward and reverse directions by the Genewiz sequencing platform (United Kingdom) and the sequences submitted to GenBank. Sequence assemblage and trimming were performed using Geneious v. 8.1.6 (final size of sequence = 255 bp). Clean sequences were aligned using MEGA 7 with 53 other sequences available in GenBank, including representatives of the four genetic lineages identified in Africa (see Appendix S1).
Phylogenetic relationship between sequences was inferred with a maximum likelihood method as implemented in MEGA 7, with node supports calculated by bootstrap analyses (1,000 replicates).

| Isolation of the virus
A total of 12 samples were selected based on RT-PCR results to attempt virus isolation with CHS-20 cells with 1% antibiotics and 10% foetal bovine serum (Invitrogen) (Adombi et al., 2011). One negative and one positive (PPRV isolate from Ivory Coast) controls were included in the virus isolation attempts. The cells were checked daily for signs of cellular cytopathic effects (CPE) under a microscope. If no CPE were observed after 1 week, cells were trypsinized and passaged in fresh medium with only 5% of foetal bovine serum. If no CPE was observed after 2 weeks following the blind passage, the virus isolation was considered negative. Cells with CPE were stored in the freezer at −80°C.

| RE SULTS AND D ISCUSS I ON
Swabs and/or tissue samples were collected from a total of 318 and 43 goats in Mali and Senegal, respectively (Table 1) (Table 1, Table S1). Many sequences obtained were identical, with a total of 15 unique partial   (Mantip et al., 2016;Souley et al., 2019;Tounkara et al., 2018;Woma et al., 2016). The sequence was placed within a cluster with 69% node support that included PPRV strains from Nigeria (Figure 2), presented in earlier studies as belonging to a sub-cluster named IV_NigB (Mantip et al., 2016;Woma et al., 2016). It is likely that lineage IV  to better understand the reason for its capacity to spread so successfully in PPR endemic countries. These results need to be confirmed by phylogenetic analysis of the complete genome of these strains. Phylogenomic analysis would also provide more information regarding the evolution of PPRV, notably lineages II and IV in West Africa Dundon et al., 2020).

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available in GenBank at https://www.ncbi.nlm.nih.gov/, accession numbers MW281010-MW281026.